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      The Ultrastructure of the Nictitating Membrane of the Little Penguin ( Eudyptula minor, Aves)

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      1 , 2 , 3
      Integrative Organismal Biology
      Oxford University Press

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          Synopsis

          The ultrastructure of the nictitating membrane in the little penguin Eudyptula minor was studied using both scanning and transmission electron microscopy to improve our understanding of the function of ocular adnexa in diving birds. Following euthanasia, eyes were enucleated and immersion fixed in Karnovsky’s fixative. The nictitating membrane and conjunctiva were embedded in araldite and semi- or ultra-thin sections were stained and photographed using compound and transmission electron microscopes, respectively. Ultrastructural dimensions were measured directly from digital photographs. Surface ultrastructure was examined using scanning electron microscopy. The transparent nictitating membrane consists of a dense stroma surrounded by epithelia on both the external (conjunctival) and internal (bulbar) surfaces. The conjunctival surface of the membrane near the leading edge is covered by microvilli, which transition to microplicae and finally to microridges in the periphery. Beneath the epithelial cells, there is a well-developed basement membrane. Scattered throughout this epithelium are a few goblet cells. The surface of the bulbar epithelium is covered by microvilli near the leading edge, which become denser peripherally. The stroma consists of densely-packed collagen fibrils, which are randomly oriented in bundles near the leading edge but are aligned in the same direction parallel with the epithelial and corneal surfaces and with the leading edge, when the membrane is extended. The ultrastructure of the nictitating membrane in the little penguin differs from other birds and its function is predominantly protective, while preserving clear vision in both water and air.

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          The structure and transparency of the cornea.

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            Retinal Ganglion Cell Topography and Spatial Resolving Power in Penguins

            Penguins are a group of flightless seabirds that exhibit numerous morphological, behavioral and ecological adaptations to their amphibious lifestyle, but little is known about the topographic organization of neurons in their retinas. In this study, we used retinal wholemounts and stereological methods to estimate the total number and topographic distribution of retinal ganglion cells in addition to an anatomical estimate of spatial resolving power in two species of penguins: the little penguin, Eudyptula minor, and the king penguin, Aptenodytes patagonicus. The total number of ganglion cells per retina was approximately 1,200,000 in the little penguin and 1,110,000 in the king penguin. The topographic distribution of retinal ganglion cells in both species revealed the presence of a prominent horizontal visual streak with steeper gradients in the little penguin. The little penguin retinas showed ganglion cell density peaks of 21,867 cells/mm 2 , affording spatial resolution in water of 17.07–17.46 cycles/degree (12.81–13.09 cycles/degree in air). In contrast, the king penguin showed a relatively lower peak density of ganglion cells of 14,222 cells/mm 2 , but – due to its larger eye – slightly higher spatial resolution in water of 20.40 cycles/degree (15.30 cycles/degree in air). In addition, we mapped the distribution of giant ganglion cells in both penguin species using Nissl-stained wholemounts. In both species, topographic mapping of this cell type revealed the presence of an area gigantocellularis with a concentric organization of isodensity contours showing a peak in the far temporal retina of approximately 70 cells/mm 2 in the little penguin and 39 cells/mm 2 in the king penguin. Giant ganglion cell densities gradually fall towards the outermost isodensity contours revealing the presence of a vertically organized streak. In the little penguin, we confirmed our cytological characterization of giant ganglion cells using immunohistochemistry for microtubule-associated protein 2. This suite of retinal specializations, which is also observed in the closely related procellariiform seabirds, affords the eyes of the little and king penguins panoramic surveillance of the horizon and motion detection in the frontal visual field.
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              D-periodic distribution of collagen type IX along cartilage fibrils

              It has recently become apparent that collagen fibrils may be composed of more than one kind of macromolecule. To explore this possibility, we developed a procedure to purify fibril fragments from 17-d embryonic chicken sternal cartilage. The fibril population obtained shows, after negative staining, a uniformity in the banding pattern and diameter similar to the fibrils in situ. Pepsin digestion of this fibril preparation releases collagen types II, IX, and XI in the proportion of 8:1:1. Rotary shadowing of the fibrils reveals a d-periodic distribution of 35-40-nm long projections, each capped with a globular domain, which resemble in form and dimensions the aminoterminal globular and collagenous domains, NC4 and COL3, of type IX collagen. The monoclonal antibody (4D6) specific for an epitope close to the amino terminal of the COL3 domain of type IX collagen bound to these projections, thus confirming their identity. Type IX collagen is therefore distributed in a regular d-periodic arrangement along cartilage fibrils, with the chondroitin sulfate chain of type IX collagen in intimate contact with the fibril.
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                Author and article information

                Journal
                Integr Org Biol
                Integr Org Biol
                iob
                Integrative Organismal Biology
                Oxford University Press
                2517-4843
                2020
                05 January 2021
                05 January 2021
                : 2
                : 1
                : obaa048
                Affiliations
                [1 ] School of Life Sciences, La Trobe University , Bundoora, VIC 3086, Australia
                [2 ] Oceans Graduate School and the Oceans Institute, The University of Western Australia , Crawley, WA 6009, Australia
                [3 ] Department of Optometry and Vision Science, University of New South Wales , Kensington, NSW 2052, Australia
                Author notes
                Author information
                http://orcid.org/0000-0001-6236-0771
                Article
                obaa048
                10.1093/iob/obaa048
                7810573
                33791581
                cd99661a-3a6a-4651-91a6-c6303c060219
                © The Author(s) 2021. Published by Oxford University Press on behalf of the Society for Integrative and Comparative Biology.

                This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.

                History
                Page count
                Pages: 9
                Funding
                Funded by: Australian Research Council, The University of Western Australia, and La Trobe University, Australia;
                Categories
                Research Article
                AcademicSubjects/SCI00960

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